Plant diversity and soil legacy independently affect the plant metabolome and induced responses following herbivory

Abstract Plant and soil biodiversity can have significant effects on herbivore resistance mediated by plant metabolites. Here, we disentangled the independent effects of plant diversity and soil legacy on constitutive and herbivore‐induced plant metabolomes of three plant species in two complementary microcosm experiments. First, we grew plants in sterile soil with three different plant diversity levels. Second, single plant species were grown on soil with different plant diversity‐induced soil legacies. We infested a subset of all plants with Spodoptera exigua larvae, a generalist leaf‐chewing herbivore, and assessed foliar and root metabolomes. Neither plant diversity nor soil legacy had significant effects on overall foliar, root, or herbivore‐induced metabolome composition. Herbivore‐induced metabolomes, however, differed from those of control plants. We detected 139 significantly regulated metabolites by comparing plants grown in monocultures with conspecifics growing in plant or soil legacy mixtures. Moreover, plant–plant and plant–soil interactions regulated 141 metabolites in herbivore‐induced plants. Taken together, plant diversity and soil legacy independently alter the concentration and induction of plant metabolites, thus affecting the plant's defensive capability. This is a first step toward disentangling plant and soil biodiversity effects on herbivore resistance, thereby improving our understanding of the mechanisms that govern ecosystem functioning.


| Plant and soil communities are linked via the plant and influence each other
In terrestrial ecosystems, aboveground and soil communities are inseparably linked via plants (Wardle et al., 2004).Such aboveground-belowground linkages determine plant diversity effects on ecosystem functioning (Eisenhauer, 2012).Plant species often harbor unique rhizosphere communities and even influence the surrounding community composition of root-associated organisms through species-specific and context-dependent organic matter inputs (Bezemer et al., 2010;van der Putten et al., 2013).Plant species-specific root exudates, for instance, can both increase and reduce soil pathogens of neighboring heterospecific plants (Steinauer et al., 2016;van de Voorde et al., 2011).These plant-induced changes in soil biota can persist over time and result in soil legacy effects (Kardol et al., 2007).
Similarly, soil biota, especially root parasites, pathogens, and herbivores as well as mutualistic symbionts can influence plant community structure and functioning via soil feedback effects (Van Der Heijden et al., 2008;van der Putten et al., 2013;Wardle et al., 2004).Soil feedback effects are considered positive when the performance or fitness of individual plants is increased and are considered negative when plant performance or fitness is reduced (Ehrenfeld et al., 2005;Kulmatiski et al., 2008;van der Putten et al., 2013).Root parasites, pathogens, and herbivores generally induce a negative soil feedback effect, for instance, by directly removing or damaging root tissues and thus reducing root uptake capabilities.Mutualistic organisms, on the other hand, induce a positive soil feedback effect on plant growth by improving soil nutrient uptake (Bardgett & van der Putten, 2014;Wardle et al., 2004), and protection against antagonists (Latz et al., 2012).The magnitude and direction of those soil feedback effects, however, is not equal for all plant species and community contexts (Cortois et al., 2016).In turn, each plant species has a specific herbivore community which can affect soil communities via herbivory, either directly via frass or indirectly via induced responses (Bardgett & Wardle, 2010).Aboveground herbivory can, for instance, positively affect soil microbial activity by inducing the release of carbon into the rhizosphere, and change arbuscular mycorrhizal colonization by reducing the carbon allocation to roots (Gehring & Whitham, 1994;Hamilton & Frank, 2001).

| Plant diversity and soil legacy can affect the plant metabolome
Recently, research on the response of plants to plant-plant interactions and soil feedbacks has been expanded beyond the common morphological and physiological traits.Plant-plant interactions, for instance, can induce shifts in foliar metabolic profiles of multiple grassland plant species, with more than 100 metabolites changing in their concentration (Scherling et al., 2010), potentially related to competition and the production of allelopathic metabolites (Fernandez et al., 2016;Treutter, 2006).Similarly, in the presence of soil biota, plants produce species-specific shoot and root metabolomes that differ from those of plants grown in sterile conditions (Ristok et al., 2019), potentially related to mycorrhization, the interaction with nematodes and priming (Conrath et al., 2006;Rivero et al., 2015;Wurst et al., 2010).The consideration of the plant metabolome, that is, the entirety of metabolites synthesized by a plant (Oliver et al., 1998) gave rise to a new discipline, eco-metabolomics, which uses metabolome analyses, or metabolomics, to illuminate the chemical mechanisms underpinning ecological and environmental processes (Peñuelas & Sardans, 2009;Peters et al., 2018).Eco-metabolomics has been employed to investigate if plants respond on a molecular level to plant community composition and soil biota diversity (Huberty et al., 2020;Ristok et al., 2019;Scherling et al., 2010).This response can entail the de-novo synthesis of compounds, changes in metabolite concentration through regulation, and differences in overall metabolome composition (often due to a change in concentrations of a large number of metabolites).Moreover, differences in plant species richness can affect plant metabolome composition, with the most pronounced metabolome differences observed in plants grown in monoculture compared to plants grown in high-diverse plant communities (Ristok, Weinhold, et al., 2023).In addition, differential selection due to growing in monocultures or plant species mixtures can select for plants with distinct metabolomes (Zuppinger-Dingley et al., 2015).Furthermore, plant-soil interactions often affect the diversity of a plant's metabolome and can exert stronger metabolomic shifts than foliar herbivory (Huberty et al., 2020).The interaction with root parasites, pathogens, and herbivores as well as mutualistic symbionts can change the concentration of primary and secondary metabolites in leaves and roots in multiple ways, for example, up-or downregulation of specific metabolites (van Dam & Heil, 2011;van der Putten et al., 2013).These responses are generally species-specific, context-dependent, and can affect subsequent biotic interactions, such as with aboveground herbivores (Bezemer & van Dam, 2005;Ristok et al., 2019;Ristok, Weinhold, et al., 2023).

| Herbivory-induced defenses can be altered by biotic interactions
Plants have evolved a plethora of indirect and direct chemical defenses to deal with attackers (Karban & Baldwin, 1997).Induced defenses, that is, changes in the concentration of metabolites following an attack by parasites, pathogens or herbivores, or after interactions with beneficial microbes (Ferlian et al., 2018) can affect the plant metabolome locally or systemically (Bezemer & van Dam, 2005).Both plant-plant interactions and plant-soil interactions can modulate the induction of defensive metabolites.
Plant-plant interactions can affect induced defenses through plant competition, which can drive the plant to either invest resources into growth or defense (Broz et al., 2010;Fernandez et al., 2016;i.e., growth-defense trade off;van Dam & Baldwin, 2001).In addition, volatile organic compounds can induce defensive responses immediately or prime for future attacks (Baldwin et al., 2006).Plantsoil interactions with microbes, nematodes, and mycorrhizal fungi cannot just induce defenses locally in roots, but also systemically in foliar tissues (van Dam & Heil, 2011).Interactions with either of these groups of soil biota can trigger up-or downregulation of specific primary metabolites, such as amino acids and sugars, or secondary metabolites, such as glucosinolates and iridoid glycosides, in aboveground and belowground plant tissues (Hol et al., 2010;Rivero et al., 2015;Wurst et al., 2010).
Taken together, both plant-plant interactions and plant-soil interactions play significant roles in modulating the plant's metabolome, thereby affecting resistance to aboveground herbivores (Ristok et al., 2019;Ristok, Weinhold, et al., 2023;van Dam & Heil, 2011).Thus far, however, not much is known about the individual impact of plant-plant interactions or plant-soil interactions within plant communities.This is likely due to the fact that plant-plant and plant-soil interactions are tightly linked in natural communities.In addition, most microcosm studies only focus on plant-soil interaction effects (see e.g., Huberty et al., 2020;Ristok et al., 2019).Here, we explicitly investigate to which extent plantplant-interactions (PPI) or plant-soil interactions (PSI) affect the metabolomes of three forb species in a similar microcosm setup.
Both the PPI and PSI experiment covered the same range of diversity levels and plant community compositions; either as assembled plant communities grown in sterile soil (PPI) or via the inoculation of sterile substrate with conditioned field soil of communities with similar plant diversity levels (PSI).This experimental setup minimized the direct effect of soil biota in the PPI experiment through the use of sterile soil, and minimized the direct effect of heterospecific plants in the PSI experiment by planting only monospecific communities.In addition, a subset of all plants was infested with larvae of the generalist herbivore Spodoptera exigua to induce defense responses.We analyzed all samples using an untargeted metabolomics approach focusing on profiling plant secondary metabolites in leaves and roots, because secondary metabolites help plants cope with their (a)biotic environment and are involved in many biotic interactions (van Dam, 2009b;van Dam & van der Meijden, 2011;Whitehead et al., 2021).We defined PPI effects as changes in a plant's secondary metabolite profile due to direct effects of heterospecific plants, such as changes in resource availability or root exudation.In comparison, we defined PSI effects as changes in a plant's secondary metabolite profile due to direct interactions with soil biota, such as bacteria, fungi, pathogens, and nematodes.We hypothesized that (1) both plant diversity and soil legacy can alter the overall plant metabolome, as well as affect the regulation of specific metabolites.In addition, we hypothesized that (2) the induced metabolomic response to herbivory is differently affected by plant diversity and soil legacy.

| Experimental design
In summer 2017, we set up a plant-plant interaction (PPI) experiment and a plant-soil interaction (PSI) experiment with three common central European grassland forb species (Geranium pratense L., Leucanthemum vulgare (Vaill.)Lam., and Ranunculus acris L.).We chose these species based on their representation in the Trait-Based Experiment of the Jena Experiment (Ebeling et al., 2014), that is, monocultures of each species, all two-species mixtures, and the three-species mixture were established (see below).Prior to each experiment, we germinated seedlings of each species from nonsterilized seeds (Rieger-Hofmann GmbH).To assure that we would use similarly developed seedlings and to account for species-specific differences in germination, we treated the seeds as follows (based on prior germination trials; data not shown): all seeds of Geranium pratense were gently scarified with sandpaper, placed in a petri dish, and treated with 3 mL 1 g/L gibberellic acid for 24 h at 7°C.The same procedure was followed for Ranunculus acris seeds, but they were treated with 0.66 g/L gibberellic acid.No treatment was necessary for Leucanthemum vulgare seeds.Following the treatment, all seeds were transferred to plastic boxes half-filled with glass beads (50 seeds per box, only one species per box).Each box was covered with a transparent lid, and seeds were watered daily with tap water.All boxes were transferred to growth chambers (CLF Plant Climatics, Percival E-36L) with a photoperiod of 16 h light at 20°C and 8 h darkness at 12°C, and 50% relative humidity.To ensure that all seedlings reached similar sizes, seeds of R. acris were prepared and moved to the growth chamber 2 weeks before those of G. pratense and L. vulgare (i.e., seeds of R. acris were left 28 days and seeds of G. pratense and L. vulgare were left 14 days in the growth chamber).

| Plant-plant interaction experiment
We conducted the plant-plant interaction experiment in a greenhouse located at the Botanical Garden Leipzig, Germany, in May 2017.We recorded an average temperature of 22.6°C and an average relative humidity of 51.6% for the time of the experiment in the greenhouse.We used 2 L microcosms (rose pot 2.0 L; Hermann Meyer KG) filled with autoclaved (twice at 134°C for 20 min) 50:50 sand-peat (Floradur B Pot Clay Medium; Floragard) mixture.We flushed each filled microcosms with water twice to remove pulsed nutrients and toxins prior to transplanting seedlings (Alphei & Scheu, 1993;Trevors, 1996).To allow for similar soil conditions between the plant-plant interaction experiment and the plant-soil interaction experiment (see below), we chose to use a commercial sand-peat mixture as it was not possible to retrieve enough soil from the field site in Jena, Germany.We established the following plant diversity levels and communities: (1) monocultures of each species, (2) the three possible two-species mixtures, and (3) the three-species mixture (Table A1).We transplanted 12 similarly developed seedlings in each microcosm, and each plant community was replicated 10 times (total number of microcosms: 70).The relative proportion among species was equal, that is, six seedlings per species in the two-species mixture and four seedlings per species in the threespecies mixture.In the two-species mixture, we transplanted the species in an alternating pattern, while we randomized the position of each seedling in the three-species mixture.All microcosms were randomly placed on tables in the greenhouse and covered with net cages to prevent unwanted herbivory.We observed no visual signs of light deficiency and thus assume that no additional variation was introduced by the net cages.We watered all microcosms three times per week and randomized the position on the tables every 7 days.
We fertilized all microcosms with 250 mL Hoagland solution, that is, "half-Hoagland solution", after 5 weeks to counteract any loss of nutrients and ensure optimal growth.After 7 weeks of growth, we harvested five microcosms per plant diversity level to assess the constitutive metabolome (see below).
The next day, we infested two randomly selected plants per species and microcosm of the remaining microcosms with three 2nd instar Spodoptera exigua larvae each.We covered and closed each plant just above the soil with an organza net to ensure that the larvae could not escape.To ensure similar development of the larvae (eggs purchased from Entocare Biologische Gewasbescherming, Wageningen, the Netherlands), we maintained a laboratory colony on artificial diet (Elvira et al., 2010) in a growth chamber (25°C, 12 h light, 45% relative humidity).After 7 days of herbivory, we harvested the remaining microcosms to assess the herbivore-induced metabolome (see below).

| Plant-soil interaction experiment
We conducted the plant-soil interaction experiment in a greenhouse located at the Botanical Garden Leipzig, Germany, in July 2017.
We recorded an average temperature of 23.5°C and an average relative humidity of 58.6% for the time of the experiment in the greenhouse.We used PVC tube microcosms (height 20 cm, diameter 10 cm, bottom closed with 250 μm mesh) filled with 1.6 L inoculated substrate and watered each microcosm twice.We prepared the inoculated substrate by mixing autoclaved (twice at 134°C for 20 min) 50:50 sand-peat (Floradur B Pot Clay Medium; Floragard) background substrate with liquid field soil inoculum 3 weeks prior to the establishment of the experiment.In June 2017 (i.e., ~7 years after the establishment of the experiment), we collected field soil from plant communities established in 2010 as part of the Trait-Based Experiment (Ebeling et al., 2014).We collected and pooled six soil cores (2 cm × 10 cm) from each plant community accounting for within-plot heterogeneity.We sieved each field soil through a 4 mm mesh and subsequently dissolved 100 g field soil in 1 L demineralized water.We then added the liquid soil inoculum to our autoclaved background substrate (10 mL liquid inoculum per 1 kg background substrate) and stored each mixture in closed-lid plastic boxes at room temperature for 3 weeks.Each substrate-inoculum mixture was thoroughly mixed three times per week and stored with an open lid for 1 h once per week.We cleaned all used instruments, that is, sieves, boxes, beakers, mixer, before and after each step with distilled water and 70% ethanol to minimize cross contamination.
We established the following inoculated substrates (hereafter, soil legacy levels): (1) monocultures of each plant species, (2) the three possible two-species mixtures, and (3) the three-species mixture (Table A2).Each soil legacy level represents the plot from the Trait-Based Experiment, we sampled the soil from.We transplanted four similarly developed seedlings per microcosm.Seedlings of plant species were only planted into soil legacy levels that also contained the respective species in the field experiment.This setup resulted in 12 unique soil legacy level-planted species combinations.
Each soil legacy level-planted species combination was replicated 10 times (total number of microcosms: 120).All microcosms were randomly placed on tables in the greenhouse and covered with net cages to prevent unwanted herbivory.We observed no visual signs of light deficiency and thus assume that no additional variation was introduced by the net cages.We watered all microcosms three times per week and randomized the position on the tables every 7 days.We fertilized all microcosms with 250 mL Hoagland solution, that is, "half-Hoagland solution", after 5 weeks to counteract any loss of nutrients and ensure optimal growth.After 7 weeks of growth, we harvested five microcosms per soil legacy level-planted species combination to assess the constitutive metabolome (see below).The next day, we infested two randomly selected plants per microcosms of the remaining microcosms with three 2nd instar Spodoptera exigua larvae each (see above).We covered and closed each plant just above the soil with an organza net to ensure that the larvae could not escape.After 7 days of herbivory, we harvested the remaining microcosms to assess the herbivore-induced metabolome (see below).

| Sampling and sample processing
After 7 weeks of growth, we harvested five microcosms per plant diversity level in the PPI experiment and five microcosms per soil legacy level-planted species combination in the PSI experiment (Tables A1 and A2).We separated the shoot and root biomass of one randomly selected plant individual per species and microcosm by cutting the plants with scissors.We washed the roots twice under tap water to remove soil particles, and then dried the samples with paper towels.This process took roughly 30 s.All shoot and root samples were then immediately stored in paper bags on dry ice to stop further metabolism.This resulted in a total of 20 shoot and 20 root samples per species and experiment.
After one additional week of herbivory (see above), we harvested the remaining five microcosms per diversity level in the PPI experiment and five microcosms per soil legacy level-planted species combination in the PSI experiment (Tables A1 and A2).We sampled the foliar tissue of one randomly selected control and one randomly selected induced plant individual per species and microcosm by cutting the plants ca. 1 cm above the ground.All samples were then immediately stored in paper bags on dry ice.This resulted in a total of 20 control and 20 induced samples per species and experiment.
In the lab, all samples were stored in a −80°C freezer, and subsequently, freeze-dried (LABCONCO FreeZone Plus 12 Liter) for 72 h.Dried samples were stored in ziplock bags filled with silica gel at room temperature until we had ground each sample to a fine homogenous powder using a ball mill (Retsch mixer mill MM 400).

| Metabolome extraction and analysis
We extracted and analyzed all samples according to Ristok et al. ( 2019) with slight changes.We extracted 20 mg dried and ground plant tissue of each sample in 1 mL of extraction buffer (methanol/50 mM acetate buffer, pH 4.8; 50/50 [v/v]).All samples were homogenized for 5 min at 30 Hz using a Retsch mixer mill MM 400, and subsequently centrifuged for 10 min at 20,000 g and 4°C.
We collected the supernatant in a 2 mL Eppendorf tube, repeated the extraction procedure with the remaining pellet, and combined both supernatants.Lastly, we centrifuged (20,000 g, 5 min, 4°C) all extracts, transferred 200 μL to an HPLC vial, and added 800 μL extraction buffer, resulting in a 1:5 dilution.
We tested for the overall differences in constitutive foliar and root metabolome as well as herbivore-induced metabolome composition among the plant diversity or soil legacy levels by calculating permutational multivariate analyses of variance using distance matrices.We log +1 transformed the metabolite intensity data to achieve multivariate normality, and used Bray-Curtis dissimilarity to calculate the distance matrices.All analyses were permuted 9999 times.We visualized the results using Partial Least Squares -Discriminant Analysis plots.We used the same approach to test for the differences in the foliar metabolome composition between control and induced plants.We calculated each analysis separately for each species and experiment.
To test for the regulation of metabolites, we calculated differential expression analyses between the monoculture treatment level and each plant diversity or soil legacy mixture level.We used the "DESeq" function provided by the "DESeq2" package with default argument structure and values.Prior to our calculation, missing values were set to zero.We defined a metabolite to be significantly upregulated when the log2 fold change was above 0.6 and the p-value below 0.05 in comparison to the control.
Conversely, we defined a metabolite to be significantly downregulated when the log2 fold change was below −0.6 and the p-value below 0.05 in comparison to the control.We used the same approach to test for the regulation of metabolites between control and induced plants.We calculated each analysis separately for each species and experiment.
Subsequently, we assigned the putative molecular formula

| Plant diversity and soil legacy effects on plant metabolomes
Neither plant diversity nor soil legacy had a significant effect on overall foliar or root metabolome composition (Table 1).However, when we compared metabolomes of plants grown in monocultures with conspecifics growing in mixtures, we discovered a total of 139 significantly up-or downregulated metabolites in both leaves and roots (Figure 1).Across both experiments, we found that more foliar than root metabolites were regulated in response to heterospecific plant-plant and plant-soil interactions in Leucanthemum vulgare (25 vs. 12) and Ranunculus acris (36 vs. 2; Figure 1).Only in Geranium pratense were the metabolites in leaves (31 regulated metabolites) and roots (33 regulated metabolites) similarly responsive to heterospecific plant-plant or plant-soil interactions.Overall, metabolites in the leaves of R. acris were most responsive, followed by roots and leaves of G. pratense, and leaves of L. vulgare.Plant-plant interactions generally up-and downregulated metabolites across all species, while plant-soil interactions mostly downregulated metabolites in leaves and roots of G. pratense, but upregulated metabolites in leaves of R. acris (Figure 1).
We found that most regulated metabolites were uniquely synthesized by a plant in response to either plant-plant or plant-soil interactions (Figure 2).This pattern was true across leaves and roots, and across plant species.The only exceptions to this pattern occurred in leaves of G. pratense and R. acris.Here, we detected metabolites that were regulated in response to both plant-plant and plant-soil interactions (Figure 2).Given that Figure 2 compares the unique and shared regulated metabolites between PPI and PSI across all plantplant interactions and plant-soil interactions, it may occur that any given enumeration in Figure 2 is lower than in Figure 1, which represents the number of regulated metabolites when a given plant is grown in a specific plant-plant interaction and specific plant-soil interaction that is different from its conspecific control.
Moreover, we observed that plants grown either in plant-plant or plant-soil interaction, synthesized and regulated unique metabolites in leaves and roots (Figure A1).The regulated metabolites that we could tentatively assign a molecular formula and compound class or name to, mostly belonged to phenolics, in particular flavonoids, their precursors, and derivatives (Table 2).TA B L E 1 Differences in the species-specific foliar, root, and induced metabolome composition among the diversity/soil legacy levels..096

Induced metabolome composition
Note: Statistical parameters resulting from a permutational multivariate analysis of variance using distance matrices.We used Bray-Curtis dissimilarity matrices and 9999 permutations.Abbreviations: F, pseudo-F-value; p, p-value.

| Plant diversity and soil legacy effects on herbivore-induced responses.
Both in the PPI (Figure 3a-c) and the PSI (Figure 3d-f) experiment, we discovered significant differences in the foliar metabolome composition across all plant diversity levels and soil legacies between control and herbivore-induced plants in all plant species.When we tested for the regulation of metabolites between control and induced plants, we found that the total number of upregulated metabolites was higher than the total number of downregulated metabolites across all species (Figure A2).Furthermore, we observed that the absolute number of regulated metabolites was highest when plants had grown in different soil legacies in the PSI experiment.This effect was strongest for L. vulgare, while R. acris showed the overall strongest response in numbers of regulated metabolites in both the PPI and PSI experiment (Figure A2).
In contrast, we found no significant effect of plant diversity in the PPI experiment and of soil legacy in the PSI experiment on the induced metabolome in either species (Table 1).However, when we compared foliar metabolomes of herbivore-induced plants grown in monocultures with conspecifics growing in mixtures, we discovered a total of 141 significantly up-or downregulated metabolites (Figure 4).Both heterospecific plant-plant and plant-soil interactions affected the induction of metabolites compared to conspecific plant-plant or plant-soil interactions.Overall, heterospecific plantplant interactions regulated more induced metabolites than plantsoil interactions in leaves of L. vulgare (26 vs. 14) and R. acris (40 vs. 24).In comparison, heterospecific plant-soil interactions had a stronger effect on the regulation of herbivore-induced metabolites in leaves of G. pratense than heterospecific plant-plant interactions (21 vs. 16; Figure 4).In R. acris, we discovered that heterospecific plant-plant and plant-soil interactions had contrasting effects on the regulation of induced metabolites.Heterospecific plant-plant interactions strongly downregulated the induction of metabolites, while plant-soil interactions strongly upregulated the induction of metabolites (Figure 4).In contrast, these modulating effects of heterospecific plant-plant or plant-soil interactions on the induction of metabolites were mostly similar or less pronounced in herbivore-induced plants of G. pratense or L. vulgare (Figure 4).Across all species and both experiments, we found no de-novo regulated metabolites in herbivore-induced plants (Figure A3); all up-and downregulated metabolites were present in control plants as well.Similar to the analysis of regulated metabolites in leaves and roots, the tentatively assigned metabolites in herbivore-induced plants mostly belonged to the family of phenolics, in particular flavonoids, their precursors, and derivatives.Besides, we tentatively assigned two metabolites in L. vulgare as an iridoid and an alkaloid glycoside (Table 2).

| DISCUSS ION
Our study highlights that both plant-plant interactions and plantsoil interactions can affect foliar and root metabolomic profiles via the regulation of specific metabolites.We showed that metabolites that were regulated in leaves differ from those in roots, and that for two of our three plant species the number of regulated metabolites in leaves was higher than in roots.These results partially confirm our first hypothesis that both plant diversity and soil legacy can alter the overall plant metabolome, as well as affect the regulation of specific metabolites.Moreover, we revealed that the herbivore-induced metabolomic response is modulated by plantplant and plant-soil interactions.This strongly suggests that the type and diversity of biotic interactions in the environment can alter induced responses to herbivores in plants.This confirms our second hypothesis that the induced metabolomic response to herbivory is differently affected by plant diversity and soil legacy.Abbreviations: eV, fragmentation energy in electron volt; MS, mass spectrometry; PPI, plant-plant interaction; PSI, plant-soil interaction; Rt, retention time in liquid chromatography in seconds.

TA B L E 2 (Continued)
Compared to previous studies that focused on plant diversity effects in a field experiment (e.g., Scherling et al., 2010) or plant-soil feedback effects (Huberty et al., 2020;e.g., Ristok et al., 2019), our study provides new insights toward disentangling plant and soil diversity effects on plant metabolomes, and thus plant-herbivore interactions.

| Plant diversity and soil legacy effects on plant metabolomes
While we did not find any overall changes in the foliar or root metabolome composition in response to plant diversity and soil legacy, we observed the unique regulation of 139 metabolites.This is in line with previous work showing that plant diversity or soil legacy can affect the regulation of foliar metabolites (Huberty et al., 2020;Scherling et al., 2010).Our study not only adds to this body of literature but also expands our knowledge by revealing that plantplant and plant-soil interactions also affect the regulation of root metabolites.
Plant-plant and plant-soil interactions can range from positive, over neutral, to negative (Barry et al., 2019;Cortois et al., 2016).
While to our knowledge no study has yet analyzed the effects of positive plant-plant interactions on plant metabolites, in particular negative plant-plant interactions, such as competition, can affect the regulation of metabolites.In our study, we detected 45 metabolites that were significantly upregulated and 36 metabolites that were significantly downregulated as a response to plant-plant interactions.This shift in regulation is potentially a consequence of competition for resources, such as light, nutrients, and water, that can force the plant to either invest resources into growth or defense, as well as affect the production of allelopathic metabolites (Fernandez et al., 2016;Treutter, 2006).Positive plant-soil interactions with mutualists, such as arbuscular mycorrhizal fungi and plant growth-promoting bacteria, that can improve nutrient uptake and protect against antagonists (Bardgett & van der Putten, 2014;Latz et al., 2012;Wardle et al., 2004), can also affect the regulation of metabolites.In our study, we detected 24 metabolites that were significantly upregulated and 34 metabolites that were significantly downregulated as a response to plant-soil interactions.This shift in regulation may be a response to mycorrhization that, for instance, can affect phenyl alcohol and vitamin associated pathways (Rivero et al., 2015), and/or a response to negative plant-soil interactions with root parasites, pathogens, and herbivores that can reduce root uptake capabilities of resources (Bardgett & van der Putten, 2014;van der Putten et al., 2013).The infection with nematodes, for instance, can affect the regulation of iridoid glycosides (Wurst et al., 2010), while the interaction among different types of soil organisms can further influence the plant metabolome and defense (Lohmann et al., 2009).
In addition to these interaction-specific effects on foliar and root metabolomes, leaves and roots have different functions and are in different abiotic and biotic environments (van Dam, 2009a).These differences are the likely reason that certain metabolite classes in our study, such as alkaloids and phenolics, show different levels of concentration among leaves and roots (Kaplan et al., 2008).
Our study confirms that plant-plant and plant-soil interactions affect the regulation of metabolites in leaves and roots.Among the regulated metabolites, we tentatively identified some as flavonoids, iridoids, and alkaloid glycosides.Flavonoids are known as physiologically active compounds, playing important roles as signals in plant-soil biota interactions, as allelochemicals in plantplant interactions, or as deterrents in plant-herbivore interactions (Treutter, 2006).Iridoids and alkaloid glycosides are known for their significant roles in plant-herbivore interactions (Bowers & Puttick, 1988;Mithöfer & Boland, 2008).
We show for the first time that the nature of the regulated metabolites is unique to the tissue and type of biotic interaction, that is, interactions with heterospecific plants and interactions with different soil biota.Hence, our results strongly suggests that plants can adjust their constitutive metabolome, in their roots and their leaves, and specifically react to their biological environment.In light of the recent support of the interaction diversity hypothesis (Whitehead et al., 2021) for the maintenance of chemical diversity, our study presents two potentially additional avenues of biotic interactions (plant-plant and plant-soil interaction) aside from plant-herbivore interactions that may explain the maintenance of chemical diversity in the plant kingdom.Moreover, our result that the constitutive metabolome in roots and leaves is uniquely shaped by interactions with heterospecific plants and interactions with different soil biota, indicates that prior biotic interaction can affect subsequent biotic interactions, such as with aboveground herbivores.

| Plant diversity and soil legacy effects on herbivore-induced responses
Based on our samples that were taken after 7 days of herbivory to assess the herbivore-induced metabolome, we also observed alterations in the herbivore-induced metabolomic response due to plant diversity and soil legacy.Together, plant-plant and plant-soil interactions regulated 82 metabolites in control plants and 141 metabolites in herbivore-induced plants.
As shown above, plant-plant interactions can modulate growth-defense trade-offs that likely vary in strength with changes in plant diversity.In mixed communities, a combination of niche complementarity but increased competition for light, as well as a reduction of herbivory by specialized herbivores via dilution effects, may lead to a higher investment of resources into growth than defense compared to monocultures (Castagneyrol et al., 2014;Eisenhauer et al., 2019;Finch & Collier, 2000;van Moorsel et al., 2018).In fact, earlier work revealed that plants growing in mixed communities invested more resources into growth than defense-related metabolites compared to plants growing in monoculture (Broz et al., 2010), potentially reducing herbivore resistance.While we did not find differences in the overall metabolome composition of herbivore-induced plants in response to increasing plant diversity, we observed induced metabolite regulation in mixed communities.Our results suggest that the identity of the neighboring plant species determines the extent and direction of the plant-plant interaction.This has potential consequences for our understanding of plant-herbivore interactions in mixed communities, but further research is needed to confirm this hypothesis.
Plant-soil interactions, on the other hand, can prepare a plant for future attack, also called priming (Conrath et al., 2006).Systemic priming in plants can occur following interactions with soil microbes, nematodes, and mycorrhizal fungi, allowing the plant to better respond to subsequent herbivory (Kaplan et al., 2008;Martinez-Medina et al., 2016).While we have not explicitly tested for priming, it may explain why the absolute number of upregulated metabolites in herbivore-induced plants (in comparison to control plants) was highest when plants had grown in different soil legacies.However, other possible mechanisms, such as systemic acquired resistance to microbial pathogens, exist that could also explain the patterns of metabolite regulation in our study (Ryals et al., 1996).
Finally, we observed differences in the regulation of herbivore-induced metabolites among our plant species.In R. acris plants, plant-plant interactions resulted in a strong downregulation of induced metabolites, while plant-soil interactions resulted in a strong upregulation of induced metabolites.The response to either type of biotic interaction was much more attenuated in G. pratense and L.
vulgare, suggesting differences in the plant species-specific adaptability which requires future research before general assumptions can be made on the effects of plant diversity versus soil legacy on herbivore resistance.
While the present experiment provides novel insights into how metabolomic profiles, and thereby herbivore resistance, respond to changes in plant and soil biodiversity, it also calls for future studies.
To allow for the comparison of plant-plant and plant-soil interactions in our study, we inoculated sterile substrate with liquid field soil inoculum from the Trait-Based Experiment (Ebeling et al., 2014) in the PSI experiment.This, however, meant that the soil biota communities were adapted and "linked" to the plot-specific plant communities and that the sand-peat mixture that was used may have created a different environment than the one the microbes were accustomed to.To fully disentangle plant from soil biodiversity effects on the plant metabolome, one would need to expose plants to artificially constructed soil communities (see e.g., de Souza et al., 2020), also including larger soil organisms (see e.g., Lohmann et al., 2009).In addition, due to space limitations in our greenhouse, we had to run the PPI experiment before the PSI experiment, which slightly affected average temperature and humidity, and we could not set up pots with plant-plant interaction and plant-soil interaction.Future studies may fully randomize their experimental design and add PPI + PSI samples.While this was not feasible in the scope of this study, it would also be important to explore the specific effects of pre-selected functional soil biota groups, such as nematodes (e.g., Bezemer et al., 2005).Moreover, future studies should explore potential shifts in growth-defense trade-offs in more detail by exploring the performance of plants and herbivores.To our knowledge, this kind of comparable experimental design to disentangle plant-plant and plant-soil interaction effects has rarely been employed (but see Kos et al., 2015) and results and conclusions can vary between studies.
In addition, future studies may mechanistically test for the effect of PPI and PSI on certain metabolite groups, such as flavonoids, iridoids, and alkaloid glycosides.Hence, we advocate for additional experiments of that kind to generate the necessary data for more reliable conclusions.

| CON CLUS ION
Taken together, the present study shows that plant and soil biodiversity trigger unique responses in the plant's metabolomic profile that modulate the induced response to herbivory.By disentangling plant diversity from soil biodiversity effects, we advance our understanding of the mechanisms that shape plant metabolomes and thus, herbivore resistance.
A PPEN D I X 1 TA B L E A 1 Overview of the experimental design of the plant-plant-interaction experiment.

(
https:// www.chemc alc.org/ mf-finder) and compound name (https:// pubch em.ncbi.nlm.nih.gov) based on the high-resolution mass-to-charge values generated by liquid chromatography quadrupole time-of-flight mass spectrometry for 95 out of 362 up-or down regulated metabolites.In cases where our search query returned multiple candidate compounds, we limited the selection to compounds with a mass difference of less than 2 ppm and a verified description in at least one plant species.

F I G U R E 1
The total number of up-and downregulated metabolites in leaves and roots of (a, d) Geranium pratense, (b, e) Leucanthemum vulgare, and (c, f) Ranunculus acris plants grown in microcosms with different neighbors (PPI) or different soil legacies (PSI).The number depicted is in comparison to the monoculture diversity/soil legacy level.Data collected as part of the plant-plant interaction (PPI) experiment are displayed in light red (up) and dark red (down).Data collected as part of the plant-soil interaction (PSI) experiment are displayed in gray (up) and black (down).Geranium, Geranium pratense; Leucanthemum, Leucanthemum vulgare; Ranunculus, Ranunculus acris.

F
The total number of metabolites in (a-c) leaves or (d-f) roots that were uniquely up-and downregulated in plants grown in microcosms with different neighbors (PPI) or different soil legacies (PSI).Metabolites uniquely regulated in the plant-plant interaction (PPI) experiment are depicted in orange.Metabolites uniquely regulated in plant-soil interaction (PSI) experiment are depicted in violet.Overlapping areas indicate the number of up-and downregulated metabolites in both experiments.The number depicted is in comparison to the monoculture diversity/soil legacy level.
We assigned the molecular formula and the putative compound name based on the high-resolution mass-to-charge values generated by liquid chromatography quadrupole time-of-flight mass spectrometry.

F
Per species Partial Least Squares -Discriminant Analysis plots of the metabolites found in the foliar metabolomes of Geranium pratense, Leucanthemum vulgare, and Ranunculus acris control or herbivore-induced plants as part of the (a-c) plant-plant interaction experiment and (d-f) plant-soil interaction experiment.Control plants are displayed in orange squares.Induced plants are displayed in violet circles.Ellipses represent the 95% confidence interval.The metabolite intensity matrix was log + 1 transformed for the purpose of data normalization.Statistical parameters resulting from a permutational multivariate analysis of variance using distance matrices.expl.var, explained variance; F, pseudo-F-value; p, p-value.

F
The total number of up-and downregulated metabolites in leaves of (a) Geranium pratense, (b) Leucanthemum vulgare, and (c) Ranunculus acris control and herbivore-induced plants grown in microcosms with different neighbors (PPI) or different soil legacies (PSI).The number depicted is in comparison to the monoculture diversity/soil legacy level.Data collected in control plants are displayed in light orange (up) and dark orange (down).Data collected in induced plants are displayed in light green (up) and dark green (down).Induced plants were infested with Spodoptera exigua larvae for 7 days prior to sampling.Geranium, Geranium pratense; Leucanthemum, Leucanthemum vulgare; PPI, plant-plant interaction experiment; PSI, plant-soil interaction experiment; Ranunculus, Ranunculus acris.
Overview of the experimental design of the plant-soil-interaction experiment.The total number of up-and downregulated metabolites in plants grown in microcosms with (a-c) different neighbors (PPI) or (d-f) different soil legacies (PSI).Metabolites uniquely regulated in leaves are depicted in orange.Metabolites uniquely regulated in roots are depicted in violet.Overlapping areas indicate the number of up-and downregulated metabolites in both tissues.The number depicted is in comparison to the monoculture diversity/soil legacy level.PPI, plant-plant interaction experiment; PSI, plant-soil interaction experiment.F I G U R E A 2 The total number of up-and downregulated metabolites in leaves of herbivore-induced plants.The plants were subjected to 7 days of frass by three 2nd instar Spodoptera exigua larvae each.The number depicted is in comparison to control plants grown in similar soil or plant diversity levels, but without herbivore damage.Data collected as part of the plant-plant interaction (PPI) experiment are displayed in light red (up) and dark red (down).Data collected as part of the plant-soil interaction (PSI) experiment are displayed in gray (up) and black (down).Geranium, Geranium pratense; Leucanthemum, Leucanthemum vulgare; Ranunculus, Ranunculus acris.F I G U R E A 3 The total number of up-and downregulated metabolites in leaves of control and herbivore-induced plants grown in microcosms with (a-c) different neighbors (PPI) or (d-f) different soil legacies (PSI).Metabolites uniquely regulated in control plants are depicted in orange.Metabolites uniquely regulated in herbivore-induced plants are depicted in violet.Overlapping areas indicate the number of up-and downregulated metabolites in both control and herbivore-induced plants.The number depicted is in comparison to the monoculture diversity/soil legacy level.PPI, plant-plant interaction experiment; PSI, plant-soil interaction experiment.
Up-and downregulated metabolites tentatively assigned in leaves and roots of Geranium pratense, Leucanthemum vulgare, and Ranunculus acris.
TA B L E 2